Total phenolic and flavonoid contents. Table 1 screens the total phenolic and flavonoid contents of GCS grown in media contaminated with Cd and Pb at various concentrations (25–150 mg kg− 1). The total phenolic and total flavonoid contents of untreated GCS as a control (1600 ± 51 mg GAE 100 g− 1 DW and 231 ± 6.6 mg CE 100 g− 1 DW, respectively) significantly increased (P < 0.01) and reached their highest levels at the highest doses of Cd (3196 ± 54 mg GAE 100 g− 1 and 590 ± 10.2 mg CE 100 g− 1 DW, respectively) and Pb (3960 ± 76 mg GAE 100 g− 1 and 522 ± 10.5 mg CE 100 g− 1 DW, respectively) by 2.0, 2.6 and 2.5, 2.3-fold, respectively. Similarly, over-expression of phenolics and flavonoids was detected at 2.48–2.50-fold and 1.5–2.0-fold, respectively, above the levels in controls in C4 weed subjected to aluminum stress35. Accumulation of antioxidant-phenolic compounds in tissues facilitates the ability of plants to tolerate and detoxify Cd and Pb stress via their metal chelating activity and/or their antioxidant activity36. The hydroxyl and carboxylic groups of the phenolic compounds assist with the binding of metals. In addition, flavonoids, as an important class of antioxidant-phenolic compounds, aid the detoxification of ROS free radicals that are induced by heavy metal stress. Rice, as a cereal plant, responds to cadmium stress by accumulating antioxidant-phenolic compounds37. An increase in phenolic compounds is correlated with increases in Cd and Pb concentrations, which suggests that de novo synthesis of soluble phenolic compounds and/or hydrolysis of conjugated phenolic compounds occurs under heavy metal stress38. In addition, the increase in the soluble phenolic compounds that are used in the lignin biosynthesis of cell walls to create physical barriers against the harmful action of heavy metals has also been reported39. From such observations, we can conclude that the total phenolic and flavonoid contents of GCS are highly sensitive markers for indicating Pb and Cd contamination in soils, even when concentrations are low.
Table 1
Total phenolic and flavonoid contents of GCS germinated under different concentrations of Cd and Pb (25 to 150 mg kg− 1).
Metal
mg kg− 1
|
Total phenolic
mg GAE 100 g− 1
DW
|
Total flavonoid
mg CE100 g− 1
DW
|
Control
|
1600 ± 51a
|
231 ± 6.6a
|
Cd 25
|
2405 ± 80b
|
342 ± 10.2b
|
Cd 50
|
2567 ± 83c
|
410 ± 11.3c
|
Cd 75
|
2867 ± 92d
|
485 ± 12.6d
|
Cd 100
|
2980 ± 65e
|
531 ± 18.1e
|
Cd 150
|
3196 ± 54f
|
590 ± 10.2f
|
Pb 25
|
2400 ± 80g
|
360 ± 12.7g
|
Pb 50
|
2587 ± 92h
|
395 ± 13.6h
|
Pb 75
|
2752 ± 88j
|
426 ± 14.8j
|
Pb 100
|
3066 ± 79k
|
473 ± 11.2k
|
Pb 150
|
3960 ± 76i
|
522 ± 10.5i
|
GAE, gallic acid equivalent; CE, catechin equivalent; Cd, cadmium; Pb, lead. |
Antioxidant activities. Most of the recently identified phenolic compounds of GCS have potent antioxidant activity (23 Abdel-Aty et al., 2019). Therefore, the antioxidant activity of GCS germinated in media contaminated with Cd and Pb was evaluated by various antioxidant assays. In Table 2, IC50 values using DPPH and ABTS methods and EC50 values using the PMC method of untreated GCS (0.0098, 0.0065, and 0.007 mg GAE mL− 1, respectively) gradually and significantly decreased (P < 0.01) and reached to their lowest values (0.0041, 0.0011 and 0.0021 mg GAE mL− 1, respectively) at highest concentrations of Cd and Pb, respectively. Low IC50 and EC50 values reflect a high antioxidant activity. Additionally, the total antioxidant activity of GCS gradually and significantly increased relative to the control levels (P < 0.01) to reach maximum levels for Cd by 6.1-, 13.0-, and 5.8-fold and for Pb by 5.9-, 14.6-, and 8.2-fold at the highest concentrations of Cd and Pb (Table 3). This increase in antioxidant activity may be attributed to increases in the concentration of antioxidant-phenolic compounds that were associated with increasing Cd and Pb concentrations in growth media. ABTS and DPPH free radicals were tested in all antioxidation reactions of organic residues that were combined with ROS radicals40,41. Therefore, the antioxidant activities of GCS could be used as potential bioindicators for Cd and Pb toxicity. In previous studies, the antioxidant activity of rice was found to increase during Cd stress, which facilitated metal chelating and enhanced Cd tolerance37. Total antioxidant activity increased from 1.2- to 1.7-fold in Malva parviflora roots and leaves under different Cd concentration treatments42. Additionally, ABTS and DPPH free radical scavenging activity as well as PMC reduction activity gradually increased in C4 weed grown under aluminum treatments35. In contrast, chickpeas grown under different heavy metals treatments had an antioxidant activity that remained lower than that of the control, according to DPPH or ABTS assays, but that slightly increased as the concentration of accumulated metal increased43.
Table 2
The antioxidant activity of the phenolic content for GCS germinated under different concentrations of Cd and Pb (25 to 150 mg kg− 1).
Metal
mg kg− 1
|
IC50 (mg GAE mL− 1)
|
EC50(mg GAE mL− 1)
|
DPPH
|
ABTS
|
PMC
|
Control
|
0.0098 ± 2.0x10− 4a
|
0.0065 ± 5.0x10− 5a
|
0.0070 ± 6.0x10− 4a
|
Cd 25
|
0.0073 ± 1.4x10− 4b
|
0.0048 ± 3.4x10− 5b
|
0.0060 ± 5.3x10− 4b
|
Cd 50
|
0.0063 ± 1.3x10− 4c
|
0.0036 ± 2.2x10− 5c
|
0.0050 ± 5.0x10− 4c
|
Cd 75
|
0.0052 ± 1.1x10− 4d
|
0.0025 ± 1.7x10− 5d
|
0.0044 ± 4.2x10− 4d
|
Cd 100
|
0.0041 ± 1.2x10− 4e
|
0.0015 ± 2.4x10− 5e
|
0.0033 ± 4.2x10− 4e
|
Cd 150
|
0.0032 ± 1.1x10− 4f
|
0.0010 ± 3.0x10− 5f
|
0.0024 ± 4.1x10− 4f
|
Pb 25
|
0.0080 ± 1.8x10− 4g
|
0.0045 ± 3.1x10− 5g
|
0.0059 ± 5.1x10− 4b
|
Pb 50
|
0.0071 ± 2.1x10− 4b
|
0.0032 ± 3.2x10− 5h
|
0.0050 ± 5.8x10− 4c
|
Pb 75
|
0.0062 ± 2.0x10− 4c
|
0.0023 ± 2.7x10− 5j
|
0.0041 ± 6.2x10− 4j
|
Pb 100
|
0.0051 ± 2.1x10− 4d
|
0.0016 ± 2.8x10− 5k
|
0.0032 ± 5.8x10− 4e
|
Pb 150
|
0.0041 ± 2.2x10− 4e
|
0.0011 ± 2.9x10− 5i
|
0.0021 ± 6.0x10− 4g
|
GAE: Gallic acid equivalent. |
Table 3
Total antioxidant activity of GCS germinated under different concentrations of Cd and Pb (25 to 150 mg kg− 1).
Metal
mg kg− 1
|
Total antioxidant activity
(Total phenolic content of 100 g DW /IC50 or / EC50)
|
DPPH
|
ABTS
|
PMC
|
Control
|
163,265.3 ± 3200a
|
246,153.8 ± 2020a
|
228,571.4 ± 4571a
|
Cd 25
|
307,397.3 ± 3143b
|
467,500.0 ± 4700b
|
374,000 ± 3680b
|
Cd 50
|
407,460.3 ± 4140c
|
713,055.5 ± 5022c
|
513,400 ± 5101c
|
Cd 75
|
551,346.2 ± 5012d
|
1,146,800 ± 9043d
|
651,591 ± 6012d
|
Cd 100
|
726,829.3 ± 6940e
|
1,198,666 ± 9110e
|
903,030 ± 6210e
|
Cd 150
|
998,750 ± 3033f
|
3,196,000 ± 9700f
|
1,331,667 ± 8312f
|
Pb 25
|
300,000.0 ± 6022b
|
533,333.3 ± 5065g
|
406,779 ± 5275g
|
Pb 50
|
364,366.2 ± 5940g
|
808,437.5 ± 5530h
|
517,400 ± 4956c
|
Pb 75
|
443,871.0 ± 5031h
|
1,196,522 ± 7300e
|
671,220 ± 5809d
|
Pb 100
|
601,176.5 ± 4540j
|
1,916,250 ± 7600j
|
958,125 ± 7632h
|
Pb 150
|
965,854 ± 6000f
|
3,600,000 ± 8456k
|
1,885,714 ± 7737j
|
Antioxidant enzymes. Cd and Pb can cause overproduction of ROS free radicals and induce oxidative damage to plant tissues. Maintaining the balance between ROS free radicals and activation of the antioxidative system under heavy metal stress is a critical protective mechanism in plants that diminishes oxidative damage in polluted tissues15. Therefore, in the present work, the potential role of the antioxidant enzymes (POD, CAT, GR, and GST) in response to oxidative stress in GCS under Cd and Pb treatments was investigated; the results are presented in Fig. 1. POD activity gradually increased (p < 0.01) with increasing of Cd and Pb concentrations till reached highest activity (355 and 451 U g− 1, respectively) compared to the control (153 U g1) (Fig. 1A). The results observed a strong correlation between the phenolic content and POD activity of GCS germinated under Cd and Pb treatments. POD is involved in removing H2O2 as a harmful ROS-free radical by oxidation of phenolic compounds44–47. Similarly, increasing Hg accumulation in garden cress shoots was correlated with an increase in POD activity and changes in total carotenoid content19. Furthermore, POD participates in the polymerization of phenolic compounds for lignin synthesis to build a barrier that protects against toxic metal ions48.
In the antioxidant defense system, CAT also stopped the hyper-accumulation of H2O2 by converting it to water and oxygen. Here, compared with control CAT activity (100 U g− 1), an increase in Cd and Pb treatment concentrations caused a significant increase (p < 0.01) in CAT activity, which peaked (250 and 190 U g− 1, respectively) at 75 mg metal kg− 1 (Fig. 1B). Similarly, CAT and POD activities were significantly enhanced in vetiver grass, Populus nigra, Cajanus cajan49–51, and Malva parviflora 42 grown in Cd- and/or Pb-contaminated soils .
In the current study, the important GSH-utilizing enzymes, GST and GR, were also evaluated. These enzymes largely participate in the efficient metabolism of ROS free radicals and their products (52 Abdulaal et al. 2017). Hence, they tightly control heavy metal-induced oxidative stress in plants. The activities of GST and GR enzymes significantly increased relative to control levels (239 and 900 U g− 1, respectively) in all Cd and Pb treatments, and the activities reached their peak at 75 metal mg kg− 1 treatments (Cd: 700 and 566 U g-1, respectively; Pb: 2,330 and 2,318 U g− 1, respectively) (Fig. 1C and D). An increase in GST activity was previously reported in pumpkin, rice, and Cicer arietinum in response to either Cd or Pb stress53,54,17. Our results revealed a strong correlation between the phenolic compound levels and GST activity of GCS under Cd and Pb treatments, suggesting that GST not only removes toxic ROS radicals but also transports the phenolic compound chelating-metal complexes to the vacuoles55. In a previous study, GR activity increased in Brassica napus leaves under Cd stress56. In contrast, GR activity decreased in Ceratophyllum demersum and mung bean seedlings in response to Cd stress57,58. Such variations in GR activity and responses may have been due to differences in plant genotypes55. In the results described above, there was a direct relationship between Cd and Pb concentrations and POD, CAT, GST, and GR activities, which indicates that these four enzymes of the antioxidant defense system could be used as bio-indicators for Cd and Pb stress. In addition, the GCS can cope with the oxidative damage induced by Pb and Cd.
Electrophoretic pattern of peroxidase activity. Figure 2 shows the electrophoretic patterns of peroxidase in GCS germinated under Cd and Pb treatments (25–150 mg kg− 1) compared with untreated controls. Two peroxidase isozymes were detected in untreated control and the intensity of these bands increased gradually with increasing Cd and Pb concentrations; moreover, two new peroxidase isozymes appeared. This observation explains why POD activity gradually increased in all Cd and Pb treatments to cope with ROS that caused stress-related damage to plant tissues.
Bioaccumulation of Cd and Pb in GCS. The contents of Cd and Pb absorbed by GCS showed a significant increase (P < 0.01) with increasing Cd and Pb at 25, 50, 75, 100, and 150 mg kg− 1 in the order of 6.0, 8.0, 11.0, 14.0, 19.0 and 7.0, 10.0, 13.5, 16.0, 21.3%, respectively (Fig. 3). Thus, GCS seem to have the ability to take up Cd and Pb from contaminated media as phytoremediator in a short germination period (6 days). A previous study reported that garden cress plants could absorb (through their shoots and roots) 48% of Pb from soil contaminated with a 300-mg kg− 1 concentration after a 30-day culture20. Another study found that garden cress reduced 100-mg kg− 1 Hg by 33% in contaminated growth media after six repeated phytoextraction processes19. Additionally, chickpea seedlings produced maximum uptake of Pb (~ 3.8%) from contaminated soil at a Pb concentration of 250 mg kg− 1 43.
Visual changes of GCS under Cd and Pb stress. During six days of germination, the GCS did not exhibit any noticeable visual changes when subjected to different concentrations of Cd and Pb, even under substantially higher concentrations of both. One could conclude that the potent antioxidant defense system of GCS could easily counter the damaging effects of Cd and Pb toxicity. Therefore, we could not determine the toxic doses of Pb and Cd on the GCS. Some plants exposed to toxic levels of Cd and Pb decrease germination and interfere with seedling physiological processes59 such as cowpea, soybean, lettuce, and sugar beet8. While parsley seedlings that grew under considerably higher Cd concentrations didn't show any signs of visual changes60.
Bio-indicators collective data. All of the tested parameters that were measured in GCS germinated under the lowest Cd and Pb concentrations (25 mg kg− 1) observed fold increase values of 1.5–2.2-fold (Table 4). At the highest Cd and Pb concentrations (150 mg kg− 1), the fold-increase values of these parameters were slightly higher (1.7–2.9-fold), but the expected substantial increase did not occur, except for total antioxidant activity (5.8–14.6-fold) (Table 4). These observations suggest that all the tested parameters of GCS responded strongly to lower concentrations of Cd and Pb and could be used to monitor these heavy metals in contaminated soils. Notably, the antioxidant activity of phenolic compounds of GCS can be considered a potent bioindicator for monitoring contaminated soils with Cd and Pb at both low and high concentrations.
Table 4
Collective data on the fold-increase among all the tested parameters at the lowest and highest concentrations of Cd and Pb in comparison to their controls.
Metal
mg kg− 1
|
Total phenolic
content
|
Total flavonoid
content
|
Fold increases in
Total antioxidant activity
|
Antioxidant enzymes
|
|
|
|
DPPH
|
ABTS
|
PMC
|
POD
|
CAT
|
GST
|
GR
|
Cd 25
|
1.5
|
1.5
|
2.0 2.0 1.6
|
1.6
|
1.5
|
2.0
|
1.8
|
Cd 150
|
2.0
|
2.6
|
6.1 13.0 5.8
|
2.2
|
2.3
|
2.8
|
2.5
|
Pb 25
|
1.5
|
1.6
|
1.8 2.2 1.8
|
1.7
|
1.5
|
1.7
|
2.0
|
Pb 150
|
2.5
|
2.3
|
5.9 14.6 8.2
|
2.9
|
1.7
|
2.2
|
2.3
|